Several lines of evidence indicate that carcinogenesis is a multi-stepped process. The complexity of the process in vivo has prompted the development of in vitro cell transformation systems so that process can be more readily examined and manipulated to dissect the various steps involved. A detailed, elegant analysis of the number and kinds of steps required for full transformation of an established cell line of Chinese hamster fibroblasts (able to make tumors in athymic mice at low inoculum and without a long latent period) indicated that the parent cell which had previously acquired an unlimited life span (UL), had to acquire three additional phenotypes in order to be fully tumorigenic. These were: anchorage independence (AI); loss of requirement for specific growth factors (GFI); and ability to escape the immune surveillance in athymic mice (ISI). Evidence suggested that point mutations as well as chromosome aberrations were responsible for the new phenotypes in that cell system. In view of these results, we propose to determine if the same or similar steps are required for human fibroblasts to become fully tumorigenic and whether such changes can be induced by carcinogens. Although not assayed for in the Chinese hamster cells, ability to bind high levels of lectin is also characteristic of fibroblasts derived from human tumors and so, this phenotype (LB) will be included in our study. By judicious use of chemical carcinogen- or radiation-induction (single and multiple exposures), followed by proper selection, it should be possible to isolate cells with such characteristics and to combine these various phenotypes within a single human cell. It will be critical to start by inducing and selecting cells with an unlimited life span (UL) as one of the first phenotypes so that subsequent selection of cells with each of the other phenotypes will be possible. We will determine the relationship between each of these phenotypes (UL, AI, GFI, LB, ISI, as well as various combinations of the phenotypes, and full tumorigenicity. (We have already determined that exposure of diploid human fibroblasts to chemical carcinogens or UV radiation induces AI in a dose-dependent manner. When these AI cells are inoculated into X-irradiated athymic mice, they produce fibrosarcomas that begin growing immediately and reach 1 cm in diameter, but regress when the immune response of the irradiated animals recovers.) We will also analyze the biochemical mechanisms underlying the phenotypes in order to relate these to the cells' ability to form non-regressing tumors in mice. The relationship between acquisition of particular phenotypes and changes in chromosome complement will be determined.